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本帖最后由 qgjin 于 2010-5-7 04:30 编辑
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PREPARATION OF MEF CULTURES
& A9 S; h+ ^9 h J5 X% K, ~1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
% w4 w5 m, ~9 p$ {% Lplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
! W7 [! t7 H2 Z1 q2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on: `/ c5 ~* Y7 e: m, w
magnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally, S( t4 c/ U: R- m5 Z. E4 C6 Y
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).+ _2 s r8 ^2 S. |
3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of
& B: G2 _+ h, F( A3 z" QMEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.- s9 \ Y% ^ j2 k) }
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%" F. k; C) N0 U" M* ~) r
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.+ z" R8 T' N7 s! _7 J1 [) v
4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in
! h7 l0 U2 Q4 UMEF growth medium at 5% CO2 and 37℃ for 24 h.3 \+ W4 V1 q9 J3 t6 \
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
1 b1 i: C1 f( a3 d4 `6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.6 H. W0 r7 q. d, X0 [' F& t) v
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
: [8 a' m/ g" v7 {8. Add trypsin solution to the culture plates and incubate for 1–2 min.. ~0 R/ ~) y! |+ P- M7 X4 @
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).0 v! H0 m2 F2 c- H
10. Freeze any MEFs not needed immediately.
3 ]2 l$ T9 i: b+ x5 X8 I-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
- T& g0 ?8 C5 q! S! N* _11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of
; M0 M0 g% Q: u$ iundifferentiated maGSCs; prepare as follows.2 T3 X( q% U% v; v. A9 U# ?) f
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
! M! Y- ]6 y9 c- M. O4 h' Q! P13. Aspirate the mitomycin C solution and wash three times with PBS.
6 d1 l, y0 e1 q14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes
5 s) p8 b( x. fat a density of 50,000–60,000 cells/ cm2.5 |# z1 M8 c' l
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
- O" J k0 t+ v2 A6 F. e3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
7 q6 s& C( p7 N/ a, U6 ^passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
